Process and system for product recovery and cell recycle

ABSTRACT

The invention is directed to a method for recovering at least one product from a fermentation broth. The invention relates to the use of a vacuum distillation vessel to recover products, such as ethanol, from a fermentation broth, where the fermentation broth comprises viable microbial biomass, and where the recovery of the product is completed in such a manner to ensure the viability of the microbial biomass. The invention provides for product recovery at an effective rate so as to prevent the accumulation of product in the fermentation broth. To ensure the viability of the microbial biomass, the invention is designed to reduce the amount of stress on the microbial biomass. By ensuring the viability of the microbial biomass, the microbial biomass may be recycled and reused in the fermentation process, which may result in an increased efficiency of the fermentation process.

CROSS REFERENCE TO A RELATED APPLICATION

The application is a continuation of U.S. application Ser. No.16/802,057 filed Feb. 26, 2020 which in turn is a continuation of U.S.Pat. No. 10,610,802 issued Apr. 7, 2020 which claims the benefit of U.S.Provisional Application No. 62/473,850 filed Mar. 20, 2017, the contentsof which are hereby incorporated by reference in their entirety.

FIELD

This invention relates to a device and associated method for recoveringat least one product from a fermentation broth. In particular, theinvention relates to the use of a vacuum distillation vessel to recoverproducts from a fermentation broth, where the fermentation brothcontains viable microbial biomass, and where the recovery of product iscompleted in such a manner where the viability of the microbial biomassis ensured.

BACKGROUND

Carbon dioxide (CO₂) accounts for about 76% of global greenhouse gasemissions from human activities, with methane (16%), nitrous oxide (6%),and fluorinated gases (2%) accounting for the balance (United StatesEnvironmental Protection Agency). The majority of CO₂ comes from theburning of fossil fuels to produce energy, although industrial andforestry practices also emit CO₂ into the atmosphere. Reduction ofgreenhouse gas emissions, particularly CO₂, is critical to halt theprogression of global warming and the accompanying shifts in climate andweather.

It has long been recognized that catalytic processes, such as theFischer-Tropsch process, may be used to convert gases containing carbondioxide (CO₂), carbon monoxide (CO), and/or hydrogen (H₂), such asindustrial waste gas or syngas, into a variety of fuels and chemicals.Recently, however, gas fermentation has emerged as an alternativeplatform for the biological fixation of such gases. In particular,C1-fixing microorganisms have been demonstrated to convert gasescontaining CO₂, CO, and/or H₂ into products such as ethanol and2,3-butanediol. The production of such products may be limited, forexample, by slow microbial growth, limited gas consumption, sensitivityto toxins, or diversion of carbon substrates into undesired by-products.

The accumulation of products can result in a reduction in the productionefficiency of the gas fermentation process. To prevent accumulation,these products must be removed at an effective rate. If not removed atan effective rate, these products may have inhibitory and/or toxiceffects on the C1-fixing microorganisms. If the products accumulate tothe point that the C1-fixing microorganisms cannot survive, then thefermentation process may have to be stopped and restarted. Prior tobeing restarted, the fermenters often require cleaning. This can be atime-consuming process.

Another pitfall commonly associated with the recovery of products is theloss of C1-fixing microorganisms through traditional recovery processes.To overcome the loss of viable C1-fixing microorganisms, filtrationmethods have been employed. However, over time, with traditionalfiltration methods, particulate matter can build up in the filter media,which can lead to a reduction in the filtrate flux, ultimately requiringcleaning and/or replacement of the filter media.

In anaerobic gas fermentation, the slow growth rate of the bacteriacombined with product inhibition creates major constraints to theproductivity of the system. Vacuum distillation at temperatures lowenough for the bacteria to survive allows selective product removal withan added cell recycle effect, removing these constraints and allowingmuch higher system productivity without the need for costly cell recyclemembranes. Accordingly, there remains a need for a system with reducedmaintenance requirements that is capable of recovering products at aneffective rate while ensuring the viability of the C1-fixingmicroorganisms.

BRIEF SUMMARY

The invention provides a process for removing ethanol from afermentation broth, the process comprising (i) flowing a fermentationbroth comprising microbial biomass and metabolites from a bioreactor toa vessel; (ii) partially vaporizing the fermentation broth to produce anethanol rich vapor stream, and an ethanol depleted liquid streamcomprising live microbial biomass; and (iii) passing the ethanoldepleted liquid stream back to the bioreactor.

In one embodiment the fermentation broth is partially vaporised at atemperature between 37° C. and 50° C. and a pressure between 40 mbar and100 mbar.

In one embodiment a viability of the microbial biomass in the ethanoldepleted liquid stream is greater than 85%.

In one embodiment the fermentation broth comprising microbial biomassand metabolites is obtained from gas fermentation of an industrial gas.Preferably the industrial gas is selected from the group consisting ofBlast furnace gas, Basic oxygen furnace gas, gasifier syngas and PSAtail gas.

In one embodiment the fermentation broth is degassed using a cyclonicdegasser at a pressure of 0.0 bar(g) to 0.5 bar(g) prior to thevaporization step, creating a degassed broth and an evolved gas stream.In certain embodiments the evolved gas stream is water scrubbed torecover product ethanol.

In one embodiment the vessel comprises a column comprising between 8 and12 theoretical distillation stages, wherein the theoretical distillationstages are provided by structured packing. In certain embodiments thefermentation broth is flowed into the vessel at a first distillationstage. In certain embodiments, the vessel further comprises a liquidreboiler provided below the theoretical distillation stages, said liquidreboiler being separated from an upper portion of the vessel by a totaltrap-out tray. Preferably the vessel is separated vertically intobetween 2 and 4 compartments in a manner where broth from multiplefermenters may be fed to the vessel without mixing.

In one embodiment the steam which drives separation is provided by aprocess in which the vapor stream is indirectly contacted with thereboiler liquid, resulting in condensation of the vapor stream andevaporation of the reboiler liquid. Preferably the vapor stream iscompressed to a pressure of between 200 mbar to 300 mbar, and thecompression is performed by multistage turbofans. In preferredembodiments between 2 and 4 turbofan stages are provided.

In one embodiment the contacting method consists of a falling filmevaporator. In embodiments wherein incomplete condensation of the vaporstream occurs and a secondary condenser is used to condense remainingvapor. The secondary condenser provides cooling through indirect contactwith cooling water or chilled water.

In one embodiment the ethanol concentration in the liquid return streamis less than 0.1% (1 g/L).

In one embodiment the liquid return stream is stored in a cooled tankprior to return to the fermenter, the cooled tank is temperaturecontrolled to between 30° C. and 37° C., and the temperature controlresults in maintenance of cell viability greater than 85%.

In one embodiment the concentrated ethanol stream is further processedin a downstream Rectification column to separate ethanol and water. Inone embodiment the bottoms of the Rectification column are recycleddirectly back as makeup liquid to the reboiler area of the Vacuumstripper.

The invention provides a device, namely, a vacuum distillation vessel,and associated method, that utilizes a vacuum distillation vessel, forrecovering at least one product from a fermentation broth. The vacuumdistillation vessel is designed for recovering at least one product froma fermentation broth, the fermentation broth being delivered from abioreactor, the vacuum distillation vessel comprising: (a) an exteriorcasing, defining an inlet for receiving the fermentation broth, thefermentation broth comprising viable microbial biomass and at least oneproduct, an outlet for transferring a product enriched stream, and anoutlet for transferring a product depleted stream, the product depletedstream comprising viable microbial biomass, the product depleted streambeing transferred to the bioreactor; and (b) a separation sectionlocated within the casing, the separation section being bounded above byan upper tray and below by a lower tray, the separation section defininga separation medium for providing a plurality of theoreticaldistillation stages; wherein the outlet for transferring the productenriched stream is elevated relative to the inlet for receiving thefermentation broth, the inlet for receiving the fermentation broth beingelevated relative to the upper tray, and the outlet for transferring theproduct depleted stream being elevated relative to the lower tray.

Preferably, the vacuum distillation vessel is capable of processing thefermentation broth at a given feed rate. The feed rate being defined asthe volume of fermentation broth per hour. The volume of fermentationbroth is the volume of fermentation broth contained in the bioreactor.In at least one embodiment, the vacuum distillation vessel is capable ofprocessing the fermentation broth at a feed rate between 0.05 and 0.5bioreactor volumes per hour. In certain embodiments, the feed rate isbetween 0.05 to 0.1, 0.05 to 0.2, 0.05 to 0.3, 0.05 to 0.4, 0.1 to 0.3,0.1 to 0.1 to 0.5, or 0.3 to 0.5 reactor volumes per hour.

In certain instances, the fermentation broth has a given residence timein the vacuum distillation vessel. The amount of time the fermentationbroth is within the vacuum distillation vessel is the amount of timebetween the moment the fermentation broth enters through the inlet forreceiving the fermentation broth, and when the fermentation broth exitsthrough the outlet for transferring the product depleted stream.Preferably, the residence time is between 0.5 and 15 minutes. In variousembodiments, the residence time is between 0.5 and 12 minutes, 0.5 and 9minutes, 0.5 and 6 minutes, 0.5 and 3 minutes, 2 and 15 minutes, 2 and12 minutes, 2 and 9 minutes, or 2 and 6 minutes. In at least oneembodiment, the residence time is less than 15 minutes, less than 12minutes, less than 9 minutes, less than 6 minutes, less than 3 minutes,less than 2 minutes, or less than 1 minute to ensure the viability ofthe microorganisms.

The invention provides for the transferring of the product depletedstream to the bioreactor through an outlet in the casing. In at leastone embodiment, the casing of the vacuum distillation vessel isconnected to the bioreactor by piping means. The product depleted streammay be passed through the piping means from the vacuum distillationvessel to the bioreactor. Preferably, the bioreactor is operated underconditions for fermentation of a C1-containing gas from an industrialprocess.

The vacuum distillation vessel is designed so as to effectively removeproduct from the fermentation broth. The vacuum distillation vesselutilizes a separation medium as part of the removal process. Theseparation medium may be any suitable material to provide adequatevapor-liquid contact.

In certain instances, the separation medium is provided such that thepressure drop over the height of the vacuum distillation vessel is lessthan 32 mbar. In certain instances, the pressure drop over the height ofthe vacuum distillation vessel is less than 30 mbar, less than 28 mbar,less than 26 mbar, less than 24 mbar, less than 22 mbar, less than 20mbar, or less than 18 mbar.

In certain instances, the separation medium is defined by a series ofdistillation trays. The distillation trays may be any suitable series ofdistillation trays to provide adequate vapor-liquid contact.

The separation section of the vacuum distillation vessel is designed toprovide a plurality of theoretical distillation stages whereby anincreasing amount of product is vaporized from the fermentation broth asthe fermentation broth passes through the distillation stages.Preferably, the separation medium provides multiple theoreticaldistillation stages. In certain embodiments, the separation mediumprovides at least 3 theoretical distillation stages, or at least 5theoretical stages, or at least 6 theoretical stages.

The vacuum distillation vessel is designed so as to ensure the viabilityof the microbial biomass. By ensuring the viability of the microbialbiomass, the product depleted stream being passed to the bioreactor maybe utilized for the gas fermentation process. Preferably, the microbialbiomass viability is maintained at a sufficiently high percentage. Incertain instances, the viability of the microbial biomass is greaterthan 80%, or greater than 85%, or greater than 90%, or greater than 95%.

The vacuum distillation vessel may be designed in such a manner that theviability of the microbial biomass is not substantially reduced whenpassed through the vacuum distillation vessel. In certain instances, theviable microbial biomass in the product depleted stream is substantiallyequal to the viable microbial biomass in the fermentation broth.Preferably, the difference between the viability of the microbialbiomass in the product depleted stream and the viability of themicrobial biomass in the fermentation broth is less than 10%. In certaininstances, the difference is between 5 and 10%. In certain instances,the difference is less than 5%.

The viability of the microbial biomass may be measured using anysuitable means. Preferably, the viability is measured using flowcytometry and a live/dead assay. In certain instances, the measurementof viability of the microbial biomass in the fermentation broth is takenfrom the fermentation broth before entering the vacuum distillationvessel. In certain instances, the measurement of viability of themicrobial biomass in the product depleted stream is taken from theproduct depleted stream leaving the vacuum distillation vessel beforethe product depleted stream is passed to the bioreactor.

In certain instances, one or more variable may be changed as a result ofthe viability measurement. Preferably, the one or more variable changedas a result of the viability measurement is selected from the groupcomprising: pressure, temperature, residence time, product concentrationin fermentation broth, steam feed rate, and separation medium.

Preferably, the product depleted stream has reduced proportions ofproduct relative to the fermentation broth so as to prevent, or at leastmitigate, accumulation of product in the fermentation broth. Bypreventing, or at least mitigating, accumulation of product in thefermentation broth the fermentation process may be continuous.Preferably, product is recovered from a continuous fermentation process.In certain instances, the product depleted stream comprises less than 1wt. % product, or less than 0.8 wt. % product, or less than 0.6 wt. %product, or less than 0.4 wt. % product or less than 0.2 wt. % productor less than 0.1 wt. % product.

The microorganisms in the bioreactor may be capable of producing avariety of different products. Preferably, one or more productsrecovered from the continuous fermentation process is a low boilingfermentation product. In certain instances, product is selected from thegroup consisting of ethanol, acetone, isopropanol, butanol, ketones,methyl ethyl ketone, acetone, 2-butanol, 1-propanol, methyl acetate,ethyl acetate, butanone, 1,3-butadiene, isoprene, and isobutene. Incertain instances, the vacuum distillation vessel is designed withspecific constraints based upon the product being produced. In certaininstances, the product produced in the bioreactor is ethanol, acetone,isopropanol, or mixtures thereof. In various instances, the productenriched stream comprises increased proportions of ethanol, acetone,isopropanol, or mixtures thereof, relative to the fermentation broth.Preferably, the vacuum distillation vessel is designed such that ethanolcan be effectively removed from the fermentation broth. In certaininstances where ethanol is produced by the microorganisms, the productenriched stream comprises increased proportions of ethanol relative tothe fermentation broth. In certain embodiments, the vacuum distillationvessel is designed such that acetone can be effectively removed from thefermentation broth. In certain instances where acetone is produced bythe microorganisms, the product enriched stream comprises increasedproportions of acetone relative to the fermentation broth. In otherembodiments, the vacuum distillation vessel is designed such thatisopropanol can be effectively removed from the fermentation broth. Incertain instances where isopropanol is produced by the microorganisms,the product enriched stream comprises increased proportions ofisopropanol relative to the fermentation broth.

These products may be further converted to produce one or more product.In at least one embodiment, at least one or more product may be furtherconverted to produce at least one component of diesel, jet fuel, and/orgasoline. In certain instances, acetone is further converted to producemethyl methacrylate. In certain instances, isopropanol is furtherconverted to produce propylene.

To effectively remove the product from the fermentation broth, whilemaintaining microorganism viability, the vacuum distillation vesseloperates at a pressure below atmospheric. Preferably, the vacuumdistillation vessel is operated at a pressure between 40 mbar(a) and 100mbar(a), or between 40 mbar(a) and 80 mbar(a), or between 40 mbar(a) and60 mbar(a), or between 50 mbar(a) and 100 mbar(a), or between 50 mbar(a)and 80 mbar(a), or between 50 mbar(a) and 70 mbar(a), or between 60mbar(a) and 100 mbar(a), or between 60 mbar(a) and 100 mbar(a), orbetween 80 mbar(a) and 100 mbar(a).

To effectively remove the product from the fermentation broth, thevacuum distillation operates at a temperature range capable of removingproduct, while ensuring the viability of the microorganisms. In certaininstances, the product is selected from the group consisting of ethanol,acetone, and isopropanol. Preferably, the vacuum distillation vessel isoperated at a temperature between 35° C. and 50° C. In one embodiment,the temperature is between 40° C. and 45° C., or between 37° C. and 45°C., or between 45° C. and 50° C. In various instances, the temperatureis less than 37° C. In embodiments designed for acetone recovery, thevacuum distillation vessel is preferably operated at a temperaturebetween 35° C. and 50° C. In certain embodiments, for acetone recovery,the temperature is between 35° C. and 45° C., or between 40° C. and 45°C., or between 45° C. and 50° C.

In certain instances, one or more by-products are produced by thefermentation. In certain instances, the one or more by-products areselected from the group consisting of carboxylic acids (i.e. acetic acidand lactic acid) and 2,3-butanediol. In certain instances, the one ormore by-products are not separated from the fermentation broth, and arereturned to the bioreactor in the product depleted stream. Due to thecontinuous return of by-products to the bioreactor, the amount ofby-product in the fermentation may accumulate. In certain instances, itis desirable to maintain the concentration of by-products in thefermentation broth below a predetermined level. The acceptableconcentration of by-products may be determined based on the tolerance ofthe microbe to the by-product. In certain instances, it may be desirableto provide the product depleted stream to a secondary separation meansto remove one or more by-product from the product depleted stream. Incertain embodiments the by-product is 2,3-butanediol and theconcentration of 2,3-butaendiol in the fermentation broth is maintainedbelow 10g/L. In certain instances, the by-product is acetic acid and theconcentration of acetic acid in the fermentation broth is maintainedbelow 10 g/L

In certain instances, the temperature of the product depleted stream iselevated such that the product depleted stream needs to be cooled priorto being passed to the bioreactor. The temperature of the stream mayhave a direct effect on the viability of the microorganism. Forinstance, higher temperatures may result in a decrease in microorganismviability. To avoid the negative effects of increased temperature, theproduct depleted stream may be cooled by any suitable cooling meansprior to being sent to the bioreactor. Preferably, the temperature ofthe product depleted stream is cooled to between 35° C. and 40° C. priorto being returned to the bioreactor. Preferably, the fermentation brothand the product depleted stream are kept below 45° C. to avoid thedetrimental effects on viability. In one embodiment, the temperature isbetween 37° C. and 45° C. to avoid detrimental effects. In certaininstances, the temperature is dependent on the microorganism being used.The effect of temperature on microorganism viability may be heightenedat higher residence times. For instance, at higher residence times, whenthe temperature is above optimal, viability of the microorganisms maydecrease.

In certain instances, the fermentation broth may contain proportions ofgas. Gas in the fermentation broth has been shown to negatively impactthe performance of the vacuum distillation vessel. This decrease inperformance may be due, at least in part, on the correlation between gasin the fermentation broth and production of foam in the vacuumdistillation vessel. To reduce the proportions of gas in thefermentation broth, a degassing vessel may be utilized. When utilizing adegassing vessel, the inlet for receiving the fermentation broth may beconnected by piping means to the degassing vessel. The degassing vesselis operated under conditions to remove at least a portion of the gasfrom the fermentation broth prior to the fermentation broth beingdelivered to the vacuum distillation vessel.

In certain instances, the degassing vessel is operated at pressure. Incertain instances, the degassing vessel is operated at any pressure lessthan the operating pressure of the bioreactor. Preferably, the degassingvessel is operated at a pressure between 0.0 bar(g) and 1.0 bar(g). Inone embodiment, the degassing vessel is operated at a pressure between0.0 bar(g) and 0.5 bar(g). Preferably, the degassing vessel removessubstantially all of the gas from the fermentation broth. In particularembodiments, the degassing vessel removes between 0 and 100% of the gasin the fermentation broth. In certain instances, the degassing vesselremoves more than 20%, more than 40%, more than 60%, or more than 80% ofthe gas from the fermentation broth. In certain instances, the degassingvessel removes at least a portion of carbon dioxide from thefermentation broth. In certain instances, the degassing vessel removesat least 20%, or at least 40%, or at least 60%, or at least 80% ofcarbon dioxide from the fermentation broth.

The vacuum distillation vessel may receive a vapor stream from areboiler. If designed to receive a vapor stream from a reboiler, theexterior casing of the vacuum distillation vessel may further define aninlet for receiving the vapor stream. This vapor stream may be producedfrom liquid from the vacuum distillation vessel. When utilizing liquidfrom the vacuum distillation vessel, the liquid may be transferred viaan outlet in the casing of the vacuum distillation vessel. Toeffectively transfer the vapor stream to the vacuum distillation vessel,the inlet for receiving the vapor stream may be located subjacentrelative to the lower tray, and the outlet for transferring the liquidstream may be located lower relative to the inlet for receiving thevapor stream.

Preferably, the liquid stream is comprised substantially of water andminimal amounts of microbial biomass. The vacuum distillation vessel isdesigned to transfer viable microbial biomass back to the bioreactor.The viable microbial biomass is contained in the product depletedstream. The vacuum distillation vessel transfers the product depleted tothe bioreactor through the outlet for transferring the product depletedstream. The outlet for transferring the product depleted stream isplaced above the lower tray. Fermentation broth, containing microbialbiomass, may pass through this lower tray. This fermentation brothpassing through may then mix with the liquid in the bottom of the vacuumdistillation vessel. Preferably, only minimal amounts of microbialbiomass end up in the liquid in the bottom of the vacuum distillationvessel. Preferably, less than 0.042 reactor volumes of the fermentationbroth, containing the microbial biomass, pass through the lower tray perhour. In certain instances, between 0.002 and 0.042 reactor volumes ofthe fermentation broth, containing the microbial biomass, pass throughthe lower tray per hour. In various embodiments, less than 0.042, lessthan 0.037, less than 0.032, less than 0.027, less than 0.022, less than0.017, less than 0.012, less than 0.007, reactor volumes of thefermentation broth, containing the microbial biomass, pass through thelower tray per hour. This liquid, including components of fermentationbroth containing microbial biomass, is then passed to the reboiler toproduce the vapor stream.

The vacuum distillation vessel may incorporate one or more additionaltrays below the lower tray. The one or more additional trays may providefor additional product removal. When including one or more additionaltrays, the fermentation broth that passes through the lower tray ispassed to the one or more additional trays where additional amounts ofproduct may be recovered. After passing through the one or moreadditional trays, the fermentation broth mixes with the liquid in thebottom of the vacuum distillation vessel. This liquid, includingcomponents of fermentation broth containing microbial biomass, is thenpassed to the reboiler to produce the vapor stream.

The vacuum distillation vessel may be separated into multiplecompartments. Preferably, when the vacuum distillation vessel isseparated into multiple compartments, the fermentation broth within eachcompartment is contained such that the fermentation broth from onecompartment does not mix with fermentation broth from anothercompartment. This separation may be achieved through any suitable means.In certain instances, the fermentation broth may be sourced frommultiple bioreactors. The product depleted stream from the fermentationbroth may be sent back to the bioreactor from which the fermentationbroth was derived. By preventing mixing between the multiplecompartments, one vacuum distillation vessel may be utilized toeffectively recover product from a plurality of bioreactors.

Preferably, the bioreactor that provides the fermentation broth isutilized for fermentation of a C1-containing substrate. ThisC1-containing substrate utilized in the fermentation process may besourced from one or more industrial processes. Preferably, theindustrial process is selected from the group comprising: carbohydratefermentation, gas fermentation, cement making, pulp and paper making,steel making, oil refining and associated processes, petrochemicalproduction, coke production, anaerobic or aerobic digestion, synthesisgas (derived from sources including but not limited to biomass, liquidwaste streams, solid waste streams, municipal streams, fossil resourcesincluding natural gas, coal and oil), natural gas extraction, oilextraction, metallurgical processes, for production and/or refinement ofaluminium, copper, and/or ferroalloys, geological reservoirs andcatalytic processes (derived from the steam sources including but notlimited to steam methane reforming, steam naphtha reforming, petroleumcoke gasification, catalyst regeneration—fluid catalyst cracking,catalyst regeneration-naphtha reforming, and dry methane reforming).

The invention provides for a method for removing at least one productfrom the fermentation broth by utilizing a vacuum distillation vessel,the method comprising: (a) passing a fermentation broth comprisingviable microbial biomass and at least one product from a bioreactor to avacuum distillation vessel; (b) partially vaporizing the fermentationbroth to produce a product enriched stream and a product depletedstream, the product depleted stream comprising viable microbial biomass;and (c) passing the product depleted stream back to the bioreactor. Theinvention may be designed in such a manner that the viability of themicrobial biomass in the fermentation broth is ensured such that, whenpassed to the bioreactor, the microbial biomass will be utilized forfermentation of a C1-containing substrate.

Preferably, the gas in the fermentation broth is monitored andcontrolled. Gas in the fermentation broth may result in a decrease inperformance of the vacuum distillation vessel. To control the gas in thefermentation broth a degassing step may be necessary. If thefermentation broth contains higher than acceptable proportions of gas,fermentation broth is passed to a degassing means prior to passing adegassed fermentation broth to the vacuum distillation vessel.

The degassing step may be completed such that an evolved gas stream isseparated from the fermentation broth, producing a degassed fermentationbroth. The degassed fermentation broth is then able to be partiallyvaporized by the vacuum distillation vessel, producing the productenriched stream and the product depleted stream.

The portion of gas that forms the evolved gas stream may containproportions of product. To prevent product loss through removal of gas,the evolved gas stream may be sent to the subsequent processing. Incertain instances, the evolved gas stream is passed to a water scrubberto recover at least one product. In certain instances, the evolved gasstream may be sent to the bioreactor.

The method may utilize a vacuum distillation vessel that comprises aseparation section located within a casing. Preferably, the separationsection located within the casing is bounded above by an upper tray andbelow by a lower tray. The separation section may provide multipletheoretical distillation stages.

The fermentation broth being processed may contain any suitablemicroorganism. For example, the microorganism may be selected from thegroup comprising: Escherichia coli, Saccharomyces cerevisiae,Clostridium acetobutylicum, Clostridium beijerinckii, Clostridiumsaccharbutyricum, Clostridium saccharoperbutylacetonicum, Clostridiumbutyricum, Clostridium diolis, Clostridium kluyveri, Clostridiumpasterianium, Clostridium novyi, Clostridium difficile, Clostridiumthermocellum, Clostridium cellulolyticum, Clostridium cellulovorans,Clostridium phytofermentans, Lactococcus lactis, Bacillus subtilis,Bacillus licheniformis, Zymomonas mobilis, Klebsiella oxytoca,Klebsiella pneumonia, Corynebacterium glutamicum, Trichoderma reesei,Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum, andMethylobacterium extorquens. In certain instances, the microorganism maybe a C1-fixing bacterium selected from the group comprising:Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta,Butyribacterium methylotrophicum, Clostridium aceticum, Clostridiumautoethanogenum, Clostridium carboxidivorans, Clostridium coskatii,Clostridium drakei, Clostridium formicoaceticum, Clostridiumljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridiumscatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorellathermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusasilvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.Preferably, the microorganism is a member of the genus Clostridium. Incertain instances, the microorganism is Clostridium autoethanogenum.

The microorganisms may be capable of producing a variety of differentproducts. Preferably, one or more products produced by themicroorganisms is a low boiling fermentation product. In certaininstances, product is selected from the group consisting of ethanol,acetone, isopropanol, butanol, ketones, methyl ethyl ketone, acetone,2-butanol, 1-propanol, methyl acetate, ethyl acetate, butanone,1,3-butadiene, isoprene, and isobutene. In certain instances, the methodis optimized based upon the product being produced. In certaininstances, the product produced in the bioreactor is ethanol.Preferably, the method is optimized such that ethanol can be effectivelyremoved from the fermentation broth. In certain instances, themicroorganism produces at least one by-product. In one embodiment the atleast one by-product is selected from the group consisting of aceticacid, lactic acid and 2,3-butanediol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram showing the vacuum distillationvessel, degassing vessel, and reboiler, in accordance with one aspect ofthe invention.

FIG. 2 is a schematic flow diagram showing the vacuum distillationvessel, degassing vessel, and reboiler, where the vacuum distillationvessel includes one or more additional trays below the lower tray, inaccordance with one aspect of the invention.

FIG. 3 is a graph showing the metabolite profile of a batch fermentationrun, in accordance with one aspect of the invention.

FIG. 4 is a graph showing the gas uptake of the batch fermentation runcorresponding with the metabolite profile shown in FIG. 3, in accordancewith one aspect of the invention.

FIG. 5 is a graph showing the viability of the microorganisms passingthrough the vacuum distillation vessel from a bioreactor with a certainconfiguration, in accordance with one aspect of the invention.

FIG. 6 is a graph showing the viability of the microorganisms passingthrough the vacuum distillation vessel from a bioreactor with adifferent configuration than that shown in FIG. 5, in accordance withone aspect of the invention.

FIG. 7 is a schematic flow diagram showing the system according to oneaspect of the invention.

DETAILED DESCRIPTION

The inventors have identified that by using a particularly designedvacuum distillation vessel, at least one product, such as ethanol, maybe effectively recovered from a fermentation broth, containing viablemicrobial biomass, while ensuring the viability of the microbialbiomass.

Definitions

The term “vacuum distillation vessel” is intended to encompass a devicefor conducting distillation under vacuum, wherein the liquid beingdistilled is enclosed at a low pressure to reduce its boiling point.Preferably, the vacuum distillation vessel includes a casing forenclosing a separation medium. Preferably, the liquid being distilled isfermentation broth comprising viable microbial biomass and at least oneproduct. Such fermentation broth may be sourced from a bioreactor. Thebioreactor may be used for fermentation of a C1-containing substrate.

“Casing” refers to the cover or shell protecting or enclosing theseparation medium. Preferably, the casing includes a number of inletsand outlets for transferring liquid and/or gas. The casing shouldinclude at least one inlet for receiving fermentation broth, at leastone outlet for transferring a product enriched stream, and at least oneoutlet for transferring a product depleted stream.

“Separation medium” is used to describe any suitable medium capable ofproviding a large surface area for vapor-liquid contact, which increasesthe effectiveness of the vacuum distillation column. Such separationmedium is designed to provide a plurality of theoretical distillationstages. In at least one embodiment, the separation medium is a series ofdistillation trays.

“Distillation trays” or “distillation plates” and the like are intendedto encompass plates and/or trays used to encourage vapor-liquid contact.Tray types include sieve, valve, and bubble cap. Sieve trays whichcontain holes for vapor to flow through are used for high capacitysituations providing high efficiency at a low cost. Although lessexpensive, valve trays, containing holes with opening and closingvalves, have the tendency to experience fouling due to the accumulationof material. Bubble cap trays contain caps and are the most advanced andexpensive of the three trays, and are highly effective in some liquidflow rate situations.

Preferably, the “upper tray” is any suitable boundary whereby thefermentation broth may be distributed downward to the separation medium.

Preferably, the “lower tray” is any suitable boundary to effectuate thetransfer of the product depleted stream through the outlet in thecasing.

A “theoretical distillation stage” is a hypothetical zone in which twophases, such as the liquid and vapor phases of a substance, establish anequilibrium with each other. The performance of many separationprocesses depends on having a series of theoretical distillation stages.The performance of a separation device, such as a vacuum distillationvessel, may be enhanced by providing an increased number of stages.Preferably, the separation medium includes a sufficient number oftheoretical distillation stages to effectively remove at least oneproduct from the fermentation broth. Preferably, the separation mediumincludes multiple theoretical distillation stages.

The term “fermentation broth” or “broth” is intended to encompass themixture of components including the nutrient media, the culture of oneor more microorganisms, and the one or more products. It should be notedthat the term microorganism and the term bacteria are usedinterchangeably throughout the document.

“Nutrient media” or “nutrient medium” is used to describe bacterialgrowth media. Generally, this term refers to a media containingnutrients and other components appropriate for the growth of a microbialculture. The term “nutrient” includes any substance that may be utilisedin a metabolic pathway of a microorganism. Exemplary nutrients includepotassium, B vitamins, trace metals and amino acids.

Preferably, the fermentation broth is sent from a “bioreactor” to thevacuum distillation vessel. The term “bioreactor” includes afermentation device consisting of one or more vessels and/or towers orpiping arrangements, which includes the Continuous Stirred Tank Reactor(CSTR), Immobilized Cell Recycles (ICR), Trickle Bed Reactor (TBR),Bubble Column, Gas Lift Fermenter, Static Mixer, a circulated loopreactor, a membrane reactor, such as a Hollow Fibre Membrane Bioreactor(HFM BR) or other vessel or other device suitable for gas-liquidcontact. The reactor is preferably adapted to receive a gaseoussubstrate comprising CO or CO₂ or H₂ or mixtures thereof. The reactormay comprise multiple reactors (stages), either in parallel or inseries. For example, the reactor may comprise a first growth reactor inwhich the bacteria are cultured and a second fermentation reactor, towhich fermentation broth from the growth reactor may be fed and in whichmost of the fermentation products may be produced.

“Gaseous substrates comprising carbon monoxide” include any gas whichcontains carbon monoxide. The gaseous substrate will typically contain asignificant proportion of CO, preferably at least about 5% to about 100%CO by volume.

While it is not necessary for the substrate to contain any hydrogen, thepresence of H₂ should not be detrimental to product formation inaccordance with methods of the invention. In particular embodiments, thepresence of hydrogen results in an improved overall efficiency ofalcohol production. For example, in particular embodiments, thesubstrate may comprise an approx. 2:1, or 1:1, or 1:2 ratio of H₂:CO. Inone embodiment, the substrate comprises about 30% or less H₂ by volume,20% or less H₂ by volume, about 15% or less H₂ by volume or about 10% orless H₂ by volume. In other embodiments, the substrate stream compriseslow concentrations of H₂, for example, less than 5%, or less than 4%, orless than 3%, or less than 2%, or less than 1%, or is substantiallyhydrogen free. The substrate may also contain some CO₂ for example, suchas about 1% to about 80% CO₂ by volume, or 1% to about 30% CO₂ byvolume. In one embodiment, the substrate comprises less than or equal toabout 20% CO₂ by volume. In particular embodiments, the substratecomprises less than or equal to about 15% CO₂ by volume, less than orequal to about 10% CO₂ by volume, less than or equal to about 5% CO₂ byvolume or substantially no CO₂.

The use of a vacuum distillation vessel with a bioreactor may increasethe efficiency of the fermentation process. The terms “increasing theefficiency”, “increased efficiency” and the like, when used in relationto a fermentation process, include, but are not limited to, increasingone or more of the rate of growth of microorganisms catalysing thefermentation, the growth and/or product production rate at elevatedproduct concentrations, the volume of desired product produced pervolume of substrate consumed, the rate of production or level ofproduction of the desired product, and the relative proportion of thedesired product produced compared with other by-products of thefermentation.

Unless the context requires otherwise, the phrases “fermenting”,“fermentation process” or “fermentation reaction” and the like, as usedherein, are intended to encompass both the growth phase and productbiosynthesis phase of the microorganisms.

The fermentation process may be described as either “batch” or“continuous”. “Batch fermentation” is used to describe a fermentationprocess where the bioreactor is filled with raw material, i.e. thecarbon source, along with microorganisms, where the products remain inthe bioreactor until fermentation is completed. In a “batch” process,after fermentation is completed, the products are extracted and thebioreactor is cleaned before the next “batch” is started. “Continuousfermentation” is used to describe a fermentation process where thefermentation process is extended for longer periods of time, and productand/or metabolite is extracted during fermentation. Preferably, thevacuum distillation vessel removes product from a “continuousfermentation” process.

A “microorganism” is a microscopic organism, especially a bacterium,archea, virus, or fungus. The microorganism of the invention istypically a bacterium. As used herein, recitation of “microorganism”should be taken to encompass “bacterium.”

“Viability” or “viability of the microbial biomass” and the like refersto the ratio of microorganisms that are alive, capable of living,developing, or reproducing to those that are not. For example, viablemicrobial biomass in a vacuum distillation vessel may refer to the ratioof live/dead microorganisms within the vacuum distillation vessel. Theinvention may be designed so that the viability of the microbial biomassis maintained at a minimum viability. In at least one embodiment, theviability of the microbial biomass is at least about 85%. In at leastone embodiment, the viable microbial biomass is returned from the vacuumdistillation vessel back to the bioreactor.

“Effective rate of product recovery” and the like refers to the rate atwhich product can be recovered from the fermentation broth so as toprevent, or at least mitigate, the toxic and/or inhibitory effectsassociated with product accumulation. The invention may be designed sothat the effective rate of product recovery is such that the viabilityof the microbial biomass is maintained above a desired threshold. Theinvention may be designed so that the level of product concentration inthe broth is kept below a desired threshold. For example, the inventionmay be designed such that the ethanol concentration in the fermentationbroth is kept below 40 g/L. In certain instances, the ethanolconcentration in the fermentation broth is kept between 25 to 35 g/L. Inparticular instances, the ethanol concentration in the fermentationbroth is less than 30 g/L, less than 35 g/L, or less than 38 g/L.Preferably, the ethanol concentration in the fermentation broth is lessthan the concentration that may result in inhibition of themicroorganism. In particular instances, the inhibition may be dependenton the microorganism being used and the product being produced.

The vacuum distillation vessel may pass the product depleted stream to a“cooling means” prior to the product depleted stream being passed to thebioreactor. The term “cooling means” may describe any suitable device orprocess capable of reducing the temperature of the product depletedstream.

The microorganisms in bioreactor may be modified from anaturally-occurring microorganism. A “parental microorganism” is amicroorganism used to generate a microorganism of the invention. Theparental microorganism may be a naturally-occurring microorganism (i.e.,a wild-type microorganism) or a microorganism that has been previouslymodified (i.e., a mutant or recombinant microorganism). Themicroorganism of the invention may be modified to express or overexpressone or more enzymes that were not expressed or overexpressed in theparental microorganism. Similarly, the microorganism of the inventionmay be modified to contain one or more genes that were not contained bythe parental microorganism. The microorganism of the invention may alsobe modified to not express or to express lower amounts of one or moreenzymes that were expressed in the parental microorganism. In oneembodiment, the parental microorganism is Clostridium autoethanogenum,Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferredembodiment, the parental microorganism is Clostridium autoethanogenumLZ1561, which was deposited on Jun. 7, 2010 with Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ) located at Inhoffenstraß7B, D-38124 Braunschwieg, Germany on Jun. 7, 2010 under the terms of theBudapest Treaty and accorded accession number DSM23693. This strain isdescribed in International Patent Application No. PCT/NZ2011/000144,which published as WO 2012/015317.

“Wood-Ljungdahl” refers to the Wood-Ljungdahl pathway of carbon fixationas described, i.e., by Ragsdale, Biochim Biophys Acta, 1784: 1873-1898,2008. “Wood-Ljungdahl microorganisms” refers, predictably, tomicroorganisms containing the Wood-Ljungdahl pathway. Generally, themicroorganism of the invention contains a native Wood-Ljungdahl pathway.Herein, a Wood-Ljungdahl pathway may be a native, unmodifiedWood-Ljungdahl pathway or it may be a Wood-Ljungdahl pathway with somedegree of genetic modification (i.e., overexpression, heterologousexpression, knockout, etc.) so long as it still functions to convert CO,CO₂, and/or H₂ to acetyl-CoA.

“C1” refers to a one-carbon molecule, for example, CO, CO₂, CH₄, orCH₃OH. “C1-oxygenate” refers to a one-carbon molecule that alsocomprises at least one oxygen atom, for example, CO, CO₂, or CH₃OH.“C1-carbon source” refers a one carbon-molecule that serves as a partialor sole carbon source for the microorganism of the invention. Forexample, a C1-carbon source may comprise one or more of CO, CO₂, CH₄,CH₃OH, or CH₂O₂. Preferably, the C1-carbon source comprises one or bothof CO and CO₂. A “C1-fixing microorganism” is a microorganism that hasthe ability to produce one or more products from a C1-carbon source.Typically, the microorganism of the invention is a C1-fixing bacterium.

An “anaerobe” is a microorganism that does not require oxygen forgrowth. An anaerobe may react negatively or even die if oxygen ispresent above a certain threshold. However, some anaerobes are capableof tolerating low levels of oxygen (i.e., 0.000001-5% oxygen).Typically, the microorganism of the invention is an anaerobe.

“Acetogens” are obligately anaerobic bacteria that use theWood-Ljungdahl pathway as their main mechanism for energy conservationand for synthesis of acetyl-CoA and acetyl-CoA-derived products, such asacetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008). Inparticular, acetogens use the Wood-Ljungdahl pathway as a (1) mechanismfor the reductive synthesis of acetyl-CoA from CO₂, (2) terminalelectron-accepting, energy conserving process, (3) mechanism for thefixation (assimilation) of CO₂ in the synthesis of cell carbon (Drake,Acetogenic Prokaryotes, In: The Prokaryotes, 3^(rd) edition, p. 354, NewYork, N.Y., 2006). All naturally occurring acetogens are C1-fixing,anaerobic, autotrophic, and non-methanotrophic. Typically, themicroorganism of the invention is an acetogen.

An “ethanologen” is a microorganism that produces or is capable ofproducing ethanol. Typically, the microorganism of the invention is anethanologen.

An “autotroph” is a microorganism capable of growing in the absence oforganic carbon. Instead, autotrophs use inorganic carbon sources, suchas CO and/or CO₂. Typically, the microorganism of the invention is anautotroph.

A “carboxydotroph” is a microorganism capable of utilizing CO as a solesource of carbon and energy. Typically, the microorganism of theinvention is a carboxydotroph.

A “methanotroph” is a microorganism capable of utilizing methane as asole source of carbon and energy. In certain embodiments, themicroorganism of the invention is a methanotroph or is derived from amethanotroph. In other embodiments, the microorganism of the inventionis not a methanotroph or is not derived from a methanotroph.

“Substrate” refers to a carbon and/or energy source for themicroorganism of the invention. Typically, the substrate is gaseous andcomprises a C1-carbon source, for example, CO, CO₂, and/or CH₄.Preferably, the substrate comprises a C1-carbon source of CO or CO+CO₂.The substrate may further comprise other non-carbon components, such asH₂, N₂, or electrons.

The term “co-substrate” refers to a substance that, while notnecessarily being the primary energy and material source for productsynthesis, can be utilised for product synthesis when added to anothersubstrate, such as the primary substrate.

Although the substrate is typically gaseous, the substrate may also beprovided in alternative forms. For example, the substrate may bedissolved in a liquid saturated with a CO-containing gas using amicrobubble dispersion generator. By way of further example, thesubstrate may be adsorbed onto a solid support.

The substrate and/or C1-carbon source may be a waste gas obtained as aby-product of an industrial process or from some other source, such asfrom automobile exhaust fumes or biomass gasification. In certainembodiments, the industrial process is selected from the groupconsisting gas emissions from carbohydrate fermentation, gasfermentation, gas emissions from cement making, pulp and paper making,steel making, oil refining and associated processes, petrochemicalproduction, coke production, anaerobic or aerobic digestion, synthesisgas (derived from sources including but not limited to biomass, liquidwaste streams, solid waste streams, municipal streams, fossil resourcesincluding natural gas, coal and oil), natural gas extraction, oilextraction, metallurgical processes, for production and/or refinement ofaluminium, copper, and/or ferroalloys, geological reservoirs, andcatalytic processes (derived from the steam sources including but notlimited to steam methane reforming, steam naphtha reforming, petroleumcoke gasification, catalyst regeneration—fluid catalyst cracking,catalyst regeneration-naphtha reforming, and dry methane reforming). Inthese embodiments, the substrate and/or C1-carbon source may be capturedfrom the industrial process before it is emitted into the atmosphere,using any convenient method.

The microorganism of the invention may be cultured with the gas streamto produce one or more products. For instance, the microorganism of theinvention may produce or may be engineered to produce ethanol (WO2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO2012/053905), butyrate (WO 2008/115080), 2,3-butanediol (WO 2009/151342and WO 2016/094334), lactate (WO 2011/112103), butene (WO 2012/024522),butadiene (WO 2012/024522), methyl ethyl ketone (2-butanone) (WO2012/024522 and WO 2013/185123), ethylene (WO 2012/026833), acetone (WO2012/115527), isopropanol (WO 2012/115527), lipids (WO 2013/036147),3-hydroxypropionate (3-HP) (WO 2013/180581), terpenes, includingisoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO2013/185123), 1,2-propanediol (WO 2014/036152), 1-propanol (WO2014/0369152), chorismate-derived products (WO 2016/191625),3-hydroxybutyrate (WO 2017/066498), and 1,3-butanediol (WO2017/0066498). In certain embodiments, microbial biomass itself may beconsidered a product.

A “native product” is a product produced by a genetically unmodifiedmicroorganism. For example, ethanol, acetate, and 2,3-butanediol arenative products of Clostridium autoethanogenum, Clostridium ljungdahlii,and Clostridium ragsdalei. A “non-native product” is a product that isproduced by a genetically modified microorganism, but is not produced bya genetically unmodified microorganism from which the geneticallymodified microorganism is derived.

“Selectivity” refers to the ratio of the production of a target productto the production of all fermentation products produced by amicroorganism. The microorganism of the invention may be engineered toproduce products at a certain selectivity or at a minimum selectivity.In one embodiment, a target product accounts for at least about 5%, 10%,15%, 20%, 30%, 50%, 75%, or 95% of all fermentation products produced bythe microorganism of the invention. In one embodiment, the targetproduct accounts for at least 10% of all fermentation products producedby the microorganism of the invention, such that the microorganism ofthe invention has a selectivity for the target product of at least 10%.In another embodiment, the target product accounts for at least 30% ofall fermentation products produced by the microorganism of theinvention, such that the microorganism of the invention has aselectivity for the target product of at least 30%.

The vacuum distillation vessel is capable of recovering one or more “lowboiling fermentation product.” A “low boiling fermentation product” is aproduct that is volatile. These products may include, but are notlimited to, ethanol, acetone, isopropanol, butanol, ketones, methylethyl ketone, 2-butanol, 1-propanol, methyl acetate, ethyl acetate,butanone, 1,3-butadiene, isoprene, and isobutene.

The culture is generally maintained in an aqueous culture medium thatcontains nutrients, vitamins, and/or minerals sufficient to permitgrowth of the microorganism. Preferably the aqueous culture medium is ananaerobic microbial growth medium, such as a minimal anaerobic microbialgrowth medium. Suitable media are well known in the art.

The culture/fermentation should desirably be carried out underappropriate conditions for production of the target product. Typically,the culture/fermentation is performed under anaerobic conditions.Reaction conditions to consider include pressure (or partial pressure),temperature, gas flow rate, liquid flow rate, media pH, media redoxpotential, agitation rate (if using a continuous stirred tank reactor),inoculum level, maximum gas substrate concentrations to ensure that gasin the liquid phase does not become limiting, and maximum productconcentrations to avoid product inhibition. In particular, the rate ofintroduction of the substrate may be controlled to ensure that theconcentration of gas in the liquid phase does not become limiting, sinceproducts may be consumed by the culture under gas-limited conditions.

Operating a bioreactor at elevated pressures allows for an increasedrate of gas mass transfer from the gas phase to the liquid phase.Accordingly, it is generally preferable to perform theculture/fermentation at pressures higher than atmospheric pressure.Also, since a given gas conversion rate is, in part, a function of thesubstrate retention time and retention time dictates the required volumeof a bioreactor, the use of pressurized systems can greatly reduce thevolume of the bioreactor required and, consequently, the capital cost ofthe culture/fermentation equipment. This, in turn, means that theretention time, defined as the liquid volume in the bioreactor dividedby the input gas flow rate, can be reduced when bioreactors aremaintained at elevated pressure rather than atmospheric pressure. Theoptimum reaction conditions will depend partly on the particularmicroorganism used. However, in general, it is preferable to operate thefermentation at a pressure higher than atmospheric pressure. Also, sincea given gas conversion rate is in part a function of substrate retentiontime and achieving a desired retention time in turn dictates therequired volume of a bioreactor, the use of pressurized systems cangreatly reduce the volume of the bioreactor required, and consequently,the capital cost of the fermentation equipment.

The term “non-naturally occurring” when used in reference to amicroorganism is intended to mean that the microorganism has at leastone genetic modification not normally found in a naturally occurringstrain of the referenced species, including wild-type strains of thereferenced species.

The terms “genetic modification,” “genetic alteration,” or “geneticengineering” broadly refer to manipulation of the genome or nucleicacids of a microorganism by the hand of man. Likewise, the terms“genetically modified,” “genetically altered,” or “geneticallyengineered” refers to a microorganism containing such a geneticmodification, genetic alteration, or genetic engineering. These termsmay be used to differentiate a lab-generated microorganism from anaturally-occurring microorganism. Methods of genetic modification ofinclude, for example, heterologous gene expression, gene or promoterinsertion or deletion, nucleic acid mutation, altered gene expression orinactivation, enzyme engineering, directed evolution, knowledge-baseddesign, random mutagenesis methods, gene shuffling, and codonoptimization.

“Recombinant” indicates that a nucleic acid, protein, or microorganismis the product of genetic modification, engineering, or recombination.Generally, the term “recombinant” refers to a nucleic acid, protein, ormicroorganism that contains or is encoded by genetic material derivedfrom multiple sources, such as two or more different strains or speciesof microorganisms. As used herein, the term “recombinant” may also beused to describe a microorganism that comprises a mutated nucleic acidor protein, including a mutated form of an endogenous nucleic acid orprotein.

“Wild type” refers to the typical form of an organism, strain, gene, orcharacteristic as it occurs in nature, as distinguished from mutant orvariant forms.

“Endogenous” refers to a nucleic acid or protein that is present orexpressed in the wild-type or parental microorganism from which themicroorganism of the invention is derived. For example, an endogenousgene is a gene that is natively present in the wild-type or parentalmicroorganism from which the microorganism of the invention is derived.In one embodiment, the expression of an endogenous gene may becontrolled by an exogenous regulatory element, such as an exogenouspromoter.

“Exogenous” refers to a nucleic acid or protein that is not present inthe wild-type or parental microorganism from which the microorganism ofthe invention is derived. In one embodiment, an exogenous gene or enzymemay be derived from a heterologous (i.e., different) strain or speciesand introduced to or expressed in the microorganism of the invention. Inanother embodiment, an exogenous gene or enzyme may be artificially orrecombinantly created and introduced to or expressed in themicroorganism of the invention. Exogenous nucleic acids may be adaptedto integrate into the genome of the microorganism of the invention or toremain in an extra-chromosomal state in the microorganism of theinvention, for example, in a plasmid.

The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,”“nucleic acid,” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide maycomprise one or more modified nucleotides, such as methylatednucleotides or nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene products.”

The terms “polypeptide”, “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein, the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

“Enzyme activity,” or simply “activity,” refers broadly to enzymaticactivity, including, but not limited, to the activity of an enzyme, theamount of an enzyme, or the availability of an enzyme to catalyze areaction. Accordingly, “increasing” enzyme activity includes increasingthe activity of an enzyme, increasing the amount of an enzyme, orincreasing the availability of an enzyme to catalyze a reaction.Similarly, “decreasing” enzyme activity includes decreasing the activityof an enzyme, decreasing the amount of an enzyme, or decreasing theavailability of an enzyme to catalyze a reaction.

“Mutated” refers to a nucleic acid or protein that has been modified inthe microorganism of the invention compared to the wild-type or parentalmicroorganism from which the microorganism of the invention is derived.In one embodiment, the mutation may be a deletion, insertion, orsubstitution in a gene encoding an enzyme. In another embodiment, themutation may be a deletion, insertion, or substitution of one or moreamino acids in an enzyme.

In particular, a “disruptive mutation” is a mutation that reduces oreliminates (i.e., “disrupts”) the expression or activity of a gene orenzyme. The disruptive mutation may partially inactivate, fullyinactivate, or delete the gene or enzyme. The disruptive mutation may bea knockout (KO) mutation. The disruptive mutation may be any mutationthat reduces, prevents, or blocks the biosynthesis of a product producedby an enzyme. The disruptive mutation may include, for example, amutation in a gene encoding an enzyme, a mutation in a geneticregulatory element involved in the expression of a gene encoding anenzyme, the introduction of a nucleic acid which produces a protein thatreduces or inhibits the activity of an enzyme, or the introduction of anucleic acid (e.g., antisense RNA, siRNA, CRISPR) or protein whichinhibits the expression of an enzyme. The disruptive mutation may beintroduced using any method known in the art.

Introduction of a disruptive mutation results in a microorganism of theinvention that produces no target product or substantially no targetproduct or a reduced amount of target product compared to the parentalmicroorganism from which the microorganism of the invention is derived.For example, the microorganism of the invention may produce no targetproduct or at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95% less target product than the parental microorganism.For example, the microorganism of the invention may produce less thanabout 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L target product.

“Codon optimization” refers to the mutation of a nucleic acid, such as agene, for optimized or improved translation of the nucleic acid in aparticular strain or species. Codon optimization may result in fastertranslation rates or higher translation accuracy. In a preferredembodiment, the genes of the invention are codon optimized forexpression in Clostridium, particularly Clostridium autoethanogenum,Clostridium ljungdahlii, or Clostridium ragsdalei. In a furtherpreferred embodiment, the genes of the invention are codon optimized forexpression in Clostridium autoethanogenum LZ1561, which is depositedunder DSMZ accession number DSM23693.

“Overexpressed” refers to an increase in expression of a nucleic acid orprotein in the microorganism of the invention compared to the wild-typeor parental microorganism from which the microorganism of the inventionis derived. Overexpression may be achieved by any means known in theart, including modifying gene copy number, gene transcription rate, genetranslation rate, or enzyme degradation rate.

The term “variants” includes nucleic acids and proteins whose sequencevaries from the sequence of a reference nucleic acid and protein, suchas a sequence of a reference nucleic acid and protein disclosed in theprior art or exemplified herein. The invention may be practiced usingvariant nucleic acids or proteins that perform substantially the samefunction as the reference nucleic acid or protein. For example, avariant protein may perform substantially the same function or catalyzesubstantially the same reaction as a reference protein. A variant genemay encode the same or substantially the same protein as a referencegene. A variant promoter may have substantially the same ability topromote the expression of one or more genes as a reference promoter.

Such nucleic acids or proteins may be referred to herein as“functionally equivalent variants.” By way of example, functionallyequivalent variants of a nucleic acid may include allelic variants,fragments of a gene, mutated genes, polymorphisms, and the like.Homologous genes from other microorganisms are also examples offunctionally equivalent variants. These include homologous genes inspecies such as Clostridium acetobutylicum, Clostridium beijerinckii, orClostridium ljungdahlii, the details of which are publicly available onwebsites such as Genbank or NCBI. Functionally equivalent variants alsoinclude nucleic acids whose sequence varies as a result of codonoptimization for a particular microorganism. A functionally equivalentvariant of a nucleic acid will preferably have at least approximately70%, approximately 80%, approximately 85%, approximately 90%,approximately 95%, approximately 98%, or greater nucleic acid sequenceidentity (percent homology) with the referenced nucleic acid. Afunctionally equivalent variant of a protein will preferably have atleast approximately 70%, approximately 80%, approximately 85%,approximately 90%, approximately 95%, approximately 98%, or greateramino acid identity (percent homology) with the referenced protein. Thefunctional equivalence of a variant nucleic acid or protein may beevaluated using any method known in the art.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. “Substantially complementary” as usedherein refers to a degree of complementarity that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids thathybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are well knownin the art (e.g., Tijssen, Laboratory techniques in biochemistry andmolecular biology-hybridization with nucleic acid probes, Second Chapter“Overview of principles of hybridization and the strategy of nucleicacid probe assay,” Elsevier, N.Y., 1993).

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

Nucleic acids may be delivered to a microorganism of the invention usingany method known in the art. For example, nucleic acids may be deliveredas naked nucleic acids or may be formulated with one or more agents,such as liposomes. The nucleic acids may be DNA, RNA, cDNA, orcombinations thereof, as is appropriate. Restriction inhibitors may beused in certain embodiments. Additional vectors may include plasmids,viruses, bacteriophages, cosmids, and artificial chromosomes. In apreferred embodiment, nucleic acids are delivered to the microorganismof the invention using a plasmid. By way of example, transformation(including transduction or transfection) may be achieved byelectroporation, ultrasonication, polyethylene glycol-mediatedtransformation, chemical or natural competence, protoplasttransformation, prophage induction, or conjugation. In certainembodiments having active restriction enzyme systems, it may benecessary to methylate a nucleic acid before introduction of the nucleicacid into a microorganism.

Furthermore, nucleic acids may be designed to comprise a regulatoryelement, such as a promoter, to increase or otherwise control expressionof a particular nucleic acid. The promoter may be a constitutivepromoter or an inducible promoter. Ideally, the promoter is aWood-Ljungdahl pathway promoter, a ferredoxin promoter, apyruvate:ferredoxin oxidoreductase promoter, an Rnf complex operonpromoter, an ATP synthase operon promoter, or aphosphotransacetylase/acetate kinase operon promoter.

A “microorganism” is a microscopic organism, especially a bacterium,archea, virus, or fungus. The microorganism of the invention istypically a bacterium. As used herein, recitation of “microorganism”should be taken to encompass “bacterium.”

A “parental microorganism” is a microorganism used to generate amicroorganism of the invention. The parental microorganism may be anaturally-occurring microorganism (i.e., a wild-type microorganism) or amicroorganism that has been previously modified (i.e., a mutant orrecombinant microorganism). The microorganism of the invention may bemodified to express or overexpress one or more enzymes that were notexpressed or overexpressed in the parental microorganism. Similarly, themicroorganism of the invention may be modified to contain one or moregenes that were not contained by the parental microorganism. Themicroorganism of the invention may also be modified to not express or toexpress lower amounts of one or more enzymes that were expressed in theparental microorganism. In one embodiment, the parental microorganism isClostridium autoethanogenum, Clostridium ljungdahlii, or Clostridiumragsdalei. In a preferred embodiment, the parental microorganism isClostridium autoethanogenum LZ1561, which was deposited on Jun. 7, 2010with Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)located at Inhoffenstraß 7B, D-38124 Braunschwieg, Germany on June 7,2010 under the terms of the Budapest Treaty and accorded accessionnumber DSM23693.

The term “derived from” indicates that a nucleic acid, protein, ormicroorganism is modified or adapted from a different (e.g., a parentalor wild-type) nucleic acid, protein, or microorganism, so as to producea new nucleic acid, protein, or microorganism. Such modifications oradaptations typically include insertion, deletion, mutation, orsubstitution of nucleic acids or genes. Generally, the microorganism ofthe invention is derived from a parental microorganism. In oneembodiment, the microorganism of the invention is derived fromClostridium autoethanogenum, Clostridium ljungdahlii, or Clostridiumragsdalei. In a preferred embodiment, the microorganism of the inventionis derived from Clostridium autoethanogenum LZ1561, which is depositedunder DSMZ accession number DSM23693.

The microorganism of the invention may be further classified based onfunctional characteristics. For example, the microorganism of theinvention may be or may be derived from a C1-fixing microorganism, ananaerobe, an acetogen, an ethanologen, a carboxydotroph, and/or amethanotroph. Table 1 provides a representative list of microorganismsand identifies their functional characteristics.

TABLE 1 C1-fixing Anaerobe Acetogen Ethanologen Autotroph CarboxydotrophMethanotroph Acetobacterium woodii + + + +/− ¹ − − − Alkalibaculumbacchii + + + + + + − Blautia producta + + + − + + − Butyribacteriummethylotrophicum + + + + + + − Clostridium aceticum + + + − + + −Clostridium autoethanogenum + + + + + + − Clostridiumcarboxidivorans + + + + + + − Clostridium coskatii + + + + + + −Clostridium drakei + + + − + + − Clostridium formicoaceticum + + + − + +− Clostridium ljungdahlii + + + + + + − Clostridium magnum + + + − + +/−² − Clostridium ragsdalei + + + + + + − Clostridium scatologenes + + +− + + − Eubacterium limosum + + + − + + − Moorellathermautotrophica + + + + + + − Moorella thermoacetica (formerly + + +  − 3 + + − Clostridium thermoaceticum) Oxobacter pfennigii + + + − + +− Sporomusa ovata + + + − + +/− ⁴ − Sporomusa silvacetica + + + − + +/−⁵ − Sporomusa sphaeroides + + + − + +/− ⁶ − Thermoanaerobacterkiuvi + + + − + − − ¹ Acetobacterium woodi can produce ethanol fromfructose, but not from gas. ² It has not been investigated whetherClostridium magnum can grow on CO. ³ One strain of Moorellathermoacetica, Moorella sp. HUC22-1, has been reported to produceethanol from gas. ⁴ It has not been investigated whether Sporomusa ovatacan grow on CO. ⁵ It has not been investigated whether Sporomusasilvacetica can grow on CO. ⁶ It has not been investigated whetherSporomusa sphaeroides can grow on CO.

In a preferred embodiment, the microorganism of the invention is derivedfrom a C1-fixing microorganism identified in Table 1. In a preferredembodiment, the microorganism of the invention is derived from anacetogen identified in Table 1. In a preferred embodiment, themicroorganism of the invention is derived from an ethanologen identifiedin Table 1. In a preferred embodiment, the microorganism of theinvention is derived from an autotroph identified in Table 1. In apreferred embodiment, the microorganism of the invention is derived froma carboxydotroph identified in Table 1. More broadly, the microorganismof the invention may be derived from any genus or species identified inTable 1.

In a preferred embodiment, the microorganism of the invention is derivedfrom the cluster of Clostridia comprising the species Clostridiumautoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.These species were first reported and characterized by Abrini, ArchMicrobiol, 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, IntJ System Bacteriol, 43: 232-236, 1993 (Clostridium ljungdahlii), andHuhnke, WO 2008/028055 (Clostridium ragsdalei).

These three species have many similarities. In particular, these speciesare all C1-fixing, anaerobic, acetogenic, ethanologenic, andcarboxydotrophic members of the genus Clostridium. These species havesimilar genotypes and phenotypes and modes of energy conservation andfermentative metabolism. Moreover, these species are clustered inclostridial rRNA homology group I with 16S rRNA DNA that is more than99% identical, have a DNA G+C content of about 22-30 mol%, aregram-positive, have similar morphology and size (logarithmic growingcells between 0.5-0.7×3-5 μm), are mesophilic (grow optimally at 30-37°C.), have similar pH ranges of about 4-7.5 (with an optimal pH of about5.5-6), lack cytochromes, and conserve energy via an Rnf complex. Also,reduction of carboxylic acids into their corresponding alcohols has beenshown in these species (Perez, Biotechnol Bioeng, 110:1066-1077, 2012).Importantly, these species also all show strong autotrophic growth onCO-containing gases, produce ethanol and acetate (or acetic acid) asmain fermentation products, and produce small amounts of 2,3-butanedioland lactic acid under certain conditions.

However, these three species also have a number of differences. Thesespecies were isolated from different sources: Clostridiumautoethanogenum from rabbit gut, Clostridium ljungdahlii from chickenyard waste, and Clostridium ragsdalei from freshwater sediment. Thesespecies differ in utilization of various sugars (e.g., rhamnose,arabinose), acids (e.g., gluconate, citrate), amino acids (e.g.,arginine, histidine), and other substrates (e.g., betaine, butanol).Moreover, these species differ in auxotrophy to certain vitamins (e.g.,thiamine, biotin). These species have differences in nucleic and aminoacid sequences of Wood-Ljungdahl pathway genes and proteins, althoughthe general organization and number of these genes and proteins has beenfound to be the same in all species (Köpke, Curr Opin Biotechnol, 22:320-325, 2011).

Thus, in summary, many of the characteristics of Clostridiumautoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei arenot specific to that species, but are rather general characteristics forthis cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, andcarboxydotrophic members of the genus Clostridium. However, since thesespecies are, in fact, distinct, the genetic modification or manipulationof one of these species may not have an identical effect in another ofthese species. For instance, differences in growth, performance, orproduct production may be observed.

The microorganism of the invention may also be derived from an isolateor mutant of Clostridium autoethanogenum, Clostridium ljungdahlii, orClostridium ragsdalei. Isolates and mutants of Clostridiumautoethanogenum include JA1-1 (DSM10061) (Abrini, Arch Microbiol, 161:345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200), and LZ1561(DSM23693). Isolates and mutants of Clostridium ljungdahlii include ATCC49587 (Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), PETCT(DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886),C-01 (ATCC 55988) (U.S. Pat. No. 6,368,819), 0-52 (ATCC 55989) (U.S.Pat. No. 6,368,819), and OTA-1 (Tirado-Acevedo, Production of bioethanolfrom synthesis gas using Clostridium ljungdahlii, PhD thesis, NorthCarolina State University, 2010). Isolates and mutants of Clostridiumragsdalei include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

The substrate generally comprises at least some amount of CO, such asabout 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol% CO. Thesubstrate may comprise a range of CO, such as about 20-80, 30-70, or40-60 mol% CO. Preferably, the substrate comprises about 40-70 mol% CO(e.g., steel mill or blast furnace gas), about 20-30 mol% CO (e.g.,basic oxygen furnace gas), or about 15-45 mol % CO (e.g., syngas). Insome embodiments, the substrate may comprise a relatively low amount ofCO, such as about 1-10 or 1-20 mol % CO. The microorganism of theinvention typically converts at least a portion of the CO in thesubstrate to a product. In some embodiments, the substrate comprises noor substantially no (<1 mol %) CO.

The substrate may comprise some amount of H₂. For example, the substratemay comprise about 1, 2, 5, 10, 15, 20, or 30 mol % H₂. In someembodiments, the substrate may comprise a relatively high amount of H₂,such as about 60, 70, 80, or 90 mol % H₂. In further embodiments, thesubstrate comprises no or substantially no (<1 mol %) H₂.

The substrate may comprise some amount of CO₂. For example, thesubstrate may comprise about 1-80 or 1-30 mol % CO₂. In someembodiments, the substrate may comprise less than about 20, 15, 10, or 5mol % CO₂. In another embodiment, the substrate comprises no orsubstantially no (<1 mol %) CO₂.

In certain embodiments, the industrial process is selected from thegroup consisting of ferrous metal products manufacturing, such as asteel mill manufacturing, non-ferrous products manufacturing, petroleumrefining, coal gasification, electric power production, carbon blackproduction, ammonia production, methanol production, and cokemanufacturing. In these embodiments, the substrate and/or C1-carbonsource may be captured from the industrial process before it is emittedinto the atmosphere, using any convenient method.

The substrate and/or C1-carbon source may be syngas, such as syngasobtained by gasification of coal or refinery residues, gasification ofbiomass or lignocellulosic material, or reforming of natural gas. Inanother embodiment, the syngas may be obtained from the gasification ofmunicipal solid waste or industrial solid waste.

The composition of the substrate may have a significant impact on theefficiency and/or cost of the reaction. For example, the presence ofoxygen (O₂) may reduce the efficiency of an anaerobic fermentationprocess. Depending on the composition of the substrate, it may bedesirable to treat, scrub, or filter the substrate to remove anyundesired impurities, such as toxins, undesired components, or dustparticles, and/or increase the concentration of desirable components.

In certain embodiments, the fermentation is performed in the absence ofcarbohydrate substrates, such as sugar, starch, lignin, cellulose, orhemicellulose.

“Increasing the efficiency,” “increased efficiency,” and the likeinclude, but are not limited to, increasing growth rate, productproduction rate or volume, product volume per volume of substrateconsumed, or product selectivity. Efficiency may be measured relative tothe performance of parental microorganism from which the microorganismof the invention is derived.

Typically, the culture is performed in a bioreactor. The term“bioreactor” includes a culture/fermentation device consisting of one ormore vessels, towers, or piping arrangements, such as a continuousstirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bedreactor (TBR), bubble column, gas lift fermenter, static mixer, or othervessel or other device suitable for gas-liquid contact. In someembodiments, the bioreactor may comprise a first growth reactor and asecond culture/fermentation reactor. The substrate may be provided toone or both of these reactors. As used herein, the terms “culture” and“fermentation” are used interchangeably. These terms encompass both thegrowth phase and product biosynthesis phase of the culture/fermentationprocess.

In certain embodiments, the fermentation is performed in the absence oflight or in the presence of an amount of light insufficient to meet theenergetic requirements of photosynthetic microorganisms. In certainembodiments, the microorganism of the invention is a non-photosyntheticmicroorganism.

Target products may be separated or purified from a fermentation brothusing any method or combination of methods known in the art, including,for example, fractional distillation, evaporation, pervaporation, gasstripping, phase separation, and extractive fermentation, including forexample, liquid-liquid extraction. In certain embodiments, targetproducts are recovered from the fermentation broth by continuouslyremoving a portion of the broth from the bioreactor, separatingmicrobial cells from the broth (conveniently by filtration), andrecovering one or more target products from the broth. Alcohols and/oracetone may be recovered, for example, by distillation. Acids may berecovered, for example, by adsorption on activated charcoal. Separatedmicrobial cells are preferably returned to the bioreactor. The cell-freepermeate remaining after target products have been removed is alsopreferably returned to the bioreactor. Additional nutrients (such as Bvitamins) may be added to the cell-free permeate to replenish the mediumbefore it is returned to the bioreactor.

Description

Energy optimized design for a commercial sized plant. This design wasoptimized for a production plant of >50 k ton/year of ethanol using gasfermentation. It was designed for minimal energy use and lowest carbonfootprint in a location with low carbon-intensive electricityproduction.

The overall distillation plant includes 3 main elements: (i) a brothstripper at low vacuum conditions with overhead vapor recompression,(ii) a ‘rectification’ column at atmospheric conditions with overheadvapor recompression; and (iii) a dehydration section to bring theethanol to fuel grade concentration. This invention focuses on theoperation of the broth stripper at low vacuum conditions.

The broth stripper section comprises; (i) a broth degasser at nearatmospheric pressure; (ii) a vacuum broth column with multiple stages ofpacking; (iii) a vapor recompression package on the broth columnoverheads (prefer multiple stage turbofans); (iv) a reboiler exchangerto transfer heat from the compressed overheads to the bottom of thecolumn (prefer falling film evaporator); (v) a trim condenser on theoverheads flow after the reboiler exchanger; and (vi) a return brothcooling tank.

Key Design Features and Reasoning:

Degasser—a Cyclonic entry degasser at 0-0.5 barg pressure. Thisminimizes foaming and fouling risks, which are typically higher risk ingas fermentation. The Vapor phase is routed to main gas fermentationplant scrubber to minimize ethanol loss. The degasser saves on aseparate scrubber and vacuum system, or making the low pressure scrubberand vacuum system much bigger.

Broth stripper column. A multiple stage packed stripping column (feed attop) is provided. Structured packing minimizes liquid residence time forbacteria to less than 5 minutes total. The multiple stage packedstripping column ideally comprises 8 to 12 theoretical stages to getethanol <0.2 wt %, or <0.1 wt %. The stripping column provides a returnbroth with a low enough ethanol concentration that a small bleed streamcan be taken without the need to strip further in a separate column.

Vacuum conditions with a bottom pressure no greater than 90 mbar, 95mbar, or 100 mbar to ensure bacterial survival. Temperature throughoutthe column depends on pressure and equilibrium ethanol concentration.Pressure drop over the packing means pressure at the top of the columnis lower. This results in a temperature gradient down the column(low→high), combined with an ethanol titer gradient (high→low). Sincethe main mechanism of cell death due to high temperature and highethanol titer are similar and additive (membrane fluidity), thisarrangement minimizes overall stress to the bacteria.

Liquid distributer designed for high rate of degassing (still bigpressure drop from degas ser with high CO₂ load). Makes use of largecolumn diameter to lower inlet degassing requirements.

Use of a water-only reboiler section with a total trap-out tray(collecting the broth) above. Broth is not subjected to the higher skintemperatures of the reboiler exchanger. Broth is not subjected to theadditional residence time of the reboiler level control volume.

All vapor to the column is generated from pure water, which requiresmakeup. This dilutes the broth return flow. However, since gasfermentation requires a bleed flow and water makeup, this is notdetrimental.

Cooling tank on broth return. Rather than pump from a level control onthe total trap-out tray within the column, a system was devised to havethe broth leave the column immediately upon hitting the tray, and entera separate vacuum vessel with a liquid seal and a pump around cooler.Level control volume required for return broth pump is now at a lower(low stress) temperature. Significantly reduces overall time at hightemperature for the bacteria

Mechanical vapor recompression (MVR) on broth stripper. Compressesoverheads vapor, allowing condensing energy to drive reboilerrequirements for the column. Compression ratio required depends oncolumn bottom temperature (affected mostly by dP over the packing), anddT required across reboiler exchanger. Main energy input for the system,so improving efficiencies here really improves overall energy efficiency(packing dP, reboiler dT). Eliminates both steam and cooling waterenergy duty requirements for the column, in exchange for approximately1/10 of these duties as electrical energy in the MVR system.

Reboiler exchanger—Uses the condensing energy of the compressed overheadvapor stream to reboil the water section at the bottom of the column.Preferred to use falling film evaporator to minimize liquid headassociated with other exchanger types. Even relatively small heights ofliquid head in the exchanger would increase pressure and thereforerequired boiling temperature on the column bottom side of the exchanger,which would increase required output pressure of the MVR (or massivelyincrease required exchanger area).

Trim condenser and vacuum scrubber—To minimize vapor flow to vacuum pumpsystem and capture as much ethanol as possible. Standard equipment.

Rectification column—Standard multistage column for splitting ethanoland water. Preferred to use packing combined with MVR system for highestefficiency. Traditional steam driven reboiler and cooling watercondenser systems are also possible. In this case energy integration ispossible for overheads: condensing energy can be used to drive the brothstripper reboiler which results in smaller MVR and reboiler exchangerrequirements, but increases the size of the trim condenser.

Dehydration system—Membrane dehydration is preferred due to more optionson heat integration. Mol sieve technology also possible.

Water integration—The water from the bottom of the rectifier can berecycled directly back to feed the water-only reboiler area at thebottom of the vacuum broth stripper. This is a combination of wateroriginally stripped in the vacuum broth stripper, and water added in thevacuum scrubber. For gas fermentation, this will include some organicacids that are stripped in the broth stripper, but they will alsovaporize again at the bottom of the broth stripper, so there will be no(or minimal) concentration factor

Vacuum distillation has been found to effectively recover product fromfermentation broth while ensuring the viability of the microorganismscontained in the fermentation broth. The fermentation broth being fed tothe vacuum distillation vessel is sourced from a bioreactor. Preferably,the bioreactor is used for fermentation of a C1-containing gaseoussubstrate. In order for the fermentation process to operatecontinuously, at least a portion of the microorganisms contained in thebroth must remain viable. These microorganisms have fairly specifictolerances to concentrations of certain products. Additionally, thesemicroorganisms have fairly specific tolerances to temperature. Forexample, in at least one embodiment, the microorganisms have an optimumgrowth temperature of 37° C. The inventors have found that by utilizingvacuum distillation, the conditions for viability are able to becontrolled in such a manner that continuous operation of thefermentation process is possible.

The vacuum distillation vessel consists of multiple elements: (1) anexterior casing defining at least one inlet for receiving fermentationbroth, one outlet for transferring a product enriched stream, and oneoutlet for transferring a product depleted stream; (2) a separationsection located within the casing, the separation section being boundedabove by an upper tray and below by a lower tray, the separation sectiondefining a separation medium for providing a plurality of theoreticaldistillation stages; and (3) a liquid level maintained at the bottom ofthe vacuum distillation vessel.

The vacuum distillation vessel is coupled with the bioreactor so as toeffectively process the fermentation broth. It was found by theinventors that by feeding the vacuum distillation vessel at a given feedrate, product accumulation in the bioreactor is controlled, therebyensuring the viability of the microorganisms. Feed rate is given interms of volumes of fermentation broth of the bioreactor per hour. Theinventors have identified that a feed rate between 0.05 and 0.5 reactorvolumes per hour allows for the broth to be effectively processed, whileensuring the viability of the microorganisms. The feed rate may bedependent, at least in part, on the vacuum distillation vesselconditions, including but not limited to, pressure, temperature,residence time, product concentration in fermentation broth, steam feedrate, and/or separation medium. In certain embodiments, the feed rate isbetween 0.05 to 0.1, 0.05 to 0.2, 0.05 to 0.3, 0.05 to 0.4, 0.1 to 0.3,0.1 to 0.1 to 0.5, or 0.3 to 0.5 reactor volumes per hour. Preferably,the feed rate is controlled such that the product depleted stream hasacceptable proportions of product.

Additionally, the inventors have identified that by keeping theresidence time, being defined as the time that the fermentation broth iswithin the vacuum distillation vessel, within a certain period of time,the viability of the microorganisms is ensured. The inventors haveidentified that a residence time between 0.5 and 15 minutes allows forthe broth to be effectively processed, while ensuring the viability ofthe microorganisms. In various embodiments, the residence time isbetween 0.5 and 12 minutes, 0.5 and 9 minutes, 0.5 and 6 minutes, 0.5and 3 minutes, 2 and 15 minutes, 2 and 12 minutes, 2 and 9 minutes, or 2and 6 minutes. In at least one embodiment, the residence time is lessthan 15 minutes, less than 12 minutes, less than 9 minutes, less than 6minutes, less than 3 minutes, less than 2 minutes, or less than 1 minuteto ensure the viability of the microorganisms.

The vacuum distillation vessel processes the fermentation broth throughuse of pressure reduction, where the pressure inside the vacuumdistillation vessel is maintained below atmospheric so as to lower thetemperature necessary to vaporize the liquid in the fermentation broth.The temperature in the vacuum distillation vessel may be dependent onthe pressure and ethanol concentration. Preferably, the liquid beingvaporized is primarily product, such as ethanol. Preferably, thepressure of the vacuum distillation vessel is maintained between 40mbar(a) and 100 mbar(a) to ensure the viability of the microorganisms.In at least one embodiment, the vacuum distillation vessel is maintainedbetween 40 mbar(a) and 80 mbar(a), between 40 mbar(a) and 90 mbar(a), orbetween 45 mbar(a) to 90 mbar(a). The pressure typically drops over theseparation medium, meaning that the pressure at the top of the vacuumdistillation vessel is lower relative to the pressure at the bottom ofthe vacuum distillation vessel. Preferably, the pressure drop over theheight of the vacuum distillation vessel is less than 32 mbar. Incertain instances, the pressure drop over the height of the vacuumdistillation vessel is less than 30 mbar, less than 28 mbar, less than26 mbar, less than 24 mbar, less than 22 mbar, less than 20 mbar, orless than 18 mbar.

This results in a temperature gradient within the vacuum distillationvessel where the temperature increases over the length of the vessel,being lowest at the top of the vacuum distillation vessel and highest atthe bottom of the vacuum distillation vessel. As the fermentation brothflows down the vacuum distillation vessel the product titer is reduced,where the product titer is highest at the top of the vacuum distillationvessel and lowest at the bottom of the vacuum distillation vessel.

The fermentation broth initially enters the vacuum distillation vesselvia an inlet in the casing. The inlet for receiving the fermentationbroth is located above the upper tray. As the fermentation broth entersthe vessel, a portion of the product in the fermentation broth isvaporized forming a product enriched stream, which rises toward the topof the vessel, exiting through an outlet in the casing. The outlet fortransferring the product enriched stream is elevated relative to theinlet for receiving the fermentation broth. The remaining fermentationbroth passes through the upper tray and through the separation medium.The separation medium provides a plurality of theoretical distillationstages. As the fermentation broth reaches each theoretical distillationstage additional product is vaporized. The vaporized product becomingpart of the product enriched stream, rising toward the top of thevessel, and exiting through an outlet in the casing. After passingthrough the separation medium, the remaining fermentation broth exitsthe vacuum distillation vessel via an outlet in the casing. Thefermentation broth exiting the casing is the product depleted stream.The product depleted stream contains viable microbial biomass. Theoutlet for transferring the product depleted stream is elevated relativeto the lower tray. The lower tray is elevated relative to the bottom ofthe vacuum distillation vessel. The bottom of the vacuum distillationvessel contains a level of liquid.

In order to increase the effectiveness of the vacuum distillation vesseland provide for the necessary vapor-liquid contact, a vapor stream maybe introduced from a reboiler to the vacuum distillation vessel via aninlet in the casing. The inlet for receiving the vapor stream is locatedsubjacent to the lower tray. The reboiler utilizes a portion of theliquid from the bottom of the vacuum distillation vessel in combinationwith energy to vaporize the liquid and create the vapor stream. Theliquid from the bottom of the vacuum distillation vessel is transferredvia an outlet in the vacuum distillation vessel casing. This outlet islocated lower than the inlet for receiving the vapor stream. The vaporstream flows upward through the separation medium, picks up portions ofproduct, and becomes part of the product enriched stream. The productenriched stream exiting through the outlet for transferring the productenriched stream. In one or more embodiment, the product enriched streammay be further processed in order to increase the concentration of theproduct.

The fermentation broth being passed to the vacuum distillation vesselmay contain proportions of gas. Gas in the fermentation broth may resultin a decrease in performance of the vacuum distillation vessel. Toprevent the performance decrease associated with gas in the fermentationbroth, a degassing vessel may be utilized. Preferably, the degassingvessel is a cyclonic degasser. Preferably, the degassing vessel isoperated at a pressure between 0.0 bar(g) and 1.0 bar(g). In oneembodiment, the degassing vessel is operated at a pressure between 0.0bar(g) and 0.5 bar(g). Preferably, the degassing vessel removessubstantially all of the gas from the fermentation broth. In particularembodiments, the degassing vessel removes between 0 and 100% of the gasin the fermentation broth. In certain instances, the degassing vesselremoves more than 20%, more than 40%, more than 60%, or more than 80% ofthe gas from the fermentation broth. The degassing vessel is operated soas to separate at least a portion of the gas from the fermentationbroth. When utilizing a cyclonic degasser, the fermentation broth isrotated, creating a low-pressure region at the center of the rotatingfermentation broth, causing the gas to separate from the fermentationbroth. The fermentation broth with reduced proportions of gas is thensent to the vacuum distillation vessel. The separated gas may containproportions of product. To recover product from the separated gas andavoid loss of product, the separated gas may be sent to a subsequentdevice and/or processing. In at least one embodiment, the separated gasmay be passed to the bioreactor.

Preferably, the product depleted stream leaving the vacuum distillationvessel is passed back to the bioreactor. The product depleted streamcontains viable microbial biomass, which, if passed back to thebioreactor, will increase the efficiency of the fermentation process.However, this product depleted stream may have a higher than optimaltemperature. Therefore, prior to being passed back to the bioreactor,the product depleted stream may undergo cooling. The cooling of theproduct depleted stream may be completed by way of a cooling means. Thecooling is conducted under conditions to reduce the temperature of theproduct depleted stream such that the product depleted streamtemperature is within an optimal range. By reducing the temperature ofthe product depleted stream prior to passing the product depleted streamto the bioreactor, unnecessary heating of the culture in the bioreactorcan be avoided. For example, if the product depleted stream were to beprovided to the bioreactor at a higher temperature relative to thefermentation broth within the bioreactor, then the recycling of theproduct depleted stream could result in a temperature increase of thefermentation broth within the bioreactor. If the temperature of thefermentation broth within the bioreactor is not maintained within anacceptable range, suitable for the microorganisms, then the viability ofthe microorganisms could decrease. Thus, monitoring and controlling thetemperature of the product depleted stream may be critical to theability of recycling the product depleted stream.

FIG. 1 shows a vacuum distillation vessel 100 for recovering at leastone product from a fermentation broth, the fermentation broth beingdelivered from a bioreactor. The vacuum distillation vessel 100comprises an exterior casing 113, defining an inlet 114 for receivingfermentation broth, an outlet 115 for transferring a product enrichedstream via piping 104, and an outlet 116 for transferring a productdepleted stream. The vacuum distillation vessel 100 also comprises aseparation section 109 located within the casing 113, the separationsection 109 is bounded above by an upper tray 112 and below by a lowertray 111. The vacuum distillation vessel 100 is designed in a way toincrease the recovery of product from the fermentation broth. The outlet115 for transferring the product enriched stream is elevated relative tothe inlet 114 for receiving the fermentation broth. The inlet 114 forreceiving the fermentation broth being elevated relative to the uppertray 112, the outlet 116 for transferring the product depleted streambeing elevated relative to the lower tray 111.

The vacuum distillation vessel 100 is designed such that the vacuumdistillation vessel 100 can process fermentation broth at a given feedrate. The feed rate is defined in terms of volume of fermentation brothin the bioreactor. Preferably, the vacuum distillation vessel 100 isdesigned such that the feed rate is between 0.05 to 0.5.

The vacuum distillation vessel 100 is designed such that thefermentation broth defines a residence time. The residence time isdefined in terms of the amount of time the fermentation broth is withinthe vacuum distillation vessel 100. The fermentation broth is deemed tobe within the vacuum distillation vessel 100 when the fermentation brothenters through the inlet 114. The fermentation broth is deemed to be outof the vacuum distillation vessel 100 when the fermentation broth exitsthrough the outlet 116. Preferably, the residence time is between 0.5and 15 minutes. In various embodiments, the residence time is between0.5 and 12 minutes, 0.5 and 9 minutes, 0.5 and 6 minutes, 0.5 and 3minutes, 2 and 15 minutes, 2 and 12 minutes, 2 and 9 minutes, or 2 and 6minutes. In at least one embodiment, the residence time is less than 15minutes, less than 12 minutes, less than 9 minutes, less than 6 minutes,less than 3 minutes, less than 2 minutes, or less than 1 minute toensure the viability of the microorganisms.

The given residence time may depend, at least in part, on the type ofseparation medium 109 within the vacuum distillation vessel 100. In atleast one embodiment, the separation medium 109 is defined by a seriesof distillation trays. Preferably, a separation medium 109 is providedsuch that a sufficient number of theoretical distillation stages areprovided to recover product. Preferably, the separation medium 109provides multiple theoretical distillation stages. In other embodiments,the separation medium 109 provides a minimum number of theoreticaldistillation stages, for example, more than 3 theoretical distillationstages, more than 4 theoretical distillation stages, more than 5theoretical distillation stages, or more than 6 theoretical distillationstages.

The vacuum distillation vessel 100 is designed so as to effectivelyrecover product in the fermentation broth and prevent productaccumulation in the bioreactor. Preferably, the product depleted streamhas reduced proportions of product such that product accumulation iseffectively reduced or eliminated. In at least one embodiment, theproduct depleted stream comprises less than 0.2 wt. % product. Incertain embodiments, the product depleted stream comprises less than 1.0wt. % product. In particular instances, the product depleted streamcomprises between 0.1 and 1.0 wt. % product. In at least one embodiment,the product being recovered is ethanol.

To effectuate the transfer of the product depleted stream, the outlet116 for transferring the product depleted stream may be connected viapiping means 102 to the bioreactor. The product depleted stream may havehigher than acceptable temperature, and thus may require cooling priorto being transferred to the bioreactor. To effectuate cooling, a coolingmeans may be provided. The cooling means may bring the product depletedstream to an acceptable temperature prior to the product depleted streambeing transferred to the bioreactor.

In some instances, the fermentation broth may have higher thanacceptable proportions of gas, and thus may require degassing prior tobeing transferred to the bioreactor. To effectuate degassing, adegassing vessel 200 may be provided. Preferably, the degassing vessel200 is a cyclonic degas ser. The degassing vessel 200 may comprise aninlet 201 for receiving the fermentation broth. This inlet 201 may beconnected via piping means 702 to the bioreactor in order to transferthe fermentation broth from the bioreactor. Preferably, the degassingvessel 200 is operated such that at least a portion of gas can beremoved from the fermentation broth prior to the fermentation brothbeing delivered to the vacuum distillation vessel 100. The degassingvessel 200 is capable of separating the gas from the fermentation brothsuch the fermentation broth is separated into an evolved gas stream anda degassed fermentation broth. The evolved gas stream exits thedegassing vessel 200 via the outlet 205. The outlet 205 may be connectedvia piping means 204 to a subsequent process to recover product from theevolved stream. In at least one embodiment, the evolved gas stream iswater scrubbed to recover product in the evolved gas stream.Additionally, the outlet 205 may be connected to the bioreactor viapiping means 204 where the evolved gas may be used in the fermentationprocess. The degassed fermentation broth is passed through an outlet 203to the vacuum distillation vessel 100 via piping means 202. In at leastone embodiment, the degassing vessel 200 is operated at a pressurebetween 0.0 bar(g) and 0.5 bar(g). In embodiments not utilizing adegassing vessel 200, the fermentation broth may be sent directly fromthe bioreactor to the inlet 114 in the vacuum distillation vessel 100via piping means 702.

The vacuum distillation vessel 100 is designed so as to ensure theviability of the microorganisms while providing product recovery.Preferably, the viability of the microorganisms in the product depletedstream is greater than 85 percent. In at least one embodiment, theviability of the microorganisms in the product depleted stream issubstantially equal to the viable microbial biomass in the incomingfermentation broth.

The vacuum distillation vessel 100 may provide for product recoverythrough use of a reboiler 800. The reboiler 800 is provided so as todirect a vapor stream to the vacuum distillation vessel 100. This vaporstream is directed through piping means 802 from the outlet 806 in thereboiler to the inlet 117 in the casing 113 of the vacuum distillationvessel 100. The vapor stream enters the vacuum distillation vessel 100and rises upward through the lower plate 111 and the separation medium109 contacting the product in the fermentation broth. The reboiler 800may create the vapor stream through use of liquid 107 located in thebottom of the vacuum distillation vessel 100. Preferably, this liquid107 is comprised substantially of water and minimal amounts of microbialbiomass. The liquid 107 may be passed through piping means 106 from anoutlet 118 in the vacuum distillation vessel 100 to an inlet 801 in thereboiler 800. In various embodiments, the liquid 107 located in thebottom of the vacuum distillation vessel 100 may be derived from anumber of sources including, but not limited to, the cooling means,steam condensate, a cogeneration unit, and/or the rectification columnbottoms.

The casing 113 of the vacuum distillation vessel 100 may comprise one ormore additional inlets 121, 119 and outlet 120 for transferring liquid107 via piping 101, 103, and 105 into and out of the vacuum distillationvessel 100. This may allow for the content and proportion of the liquid107 in the vacuum distillation vessel 100 to be controlled. In certaininstances, the piping 101, 103, and 105 may be connected to one or moreof the sources of the liquid 107.

Additionally, the vacuum distillation vessel 100 may be designed suchthat the vacuum distillation vessel 100 is separated into multiplecompartments in a manner where fermentation broth from multiplebioreactors may be passed to the vacuum distillation vessel 100 withoutmixing. This separation may be achieved through any means suitable toensure such separation.

The vacuum distillation vessel may contain one or more additional tray122 below the lower tray 111. FIG. 2 illustrates a vacuum distillationvessel 100 with additional trays 122 below the lower tray 111. Theseadditional trays 122 provide for additional product removal. The vacuumdistillation vessel 100 is designed to transfer fermentation broth,containing the viable microbial biomass, to the bioreactor through theoutlet 116, which is placed above the lower tray 111. The fermentationbroth that passes through the lower tray 111 may contain additional,albeit minimal, amounts of fermentation broth containing the viablemicrobial biomass. The fermentation broth that passes through the lowertray 111 is not passed to the bioreactor. This fermentation broth isinstead passed through the one or more additional trays 122 whereadditional product is recovered from the fermentation broth. Afterpassing through the one or more additional trays 122, the fermentationbroth mixes with the liquid 107 located in the bottom of the vacuumdistillation vessel 100. This liquid 107, including portions offermentation broth containing microbial biomass, is then passed to thereboiler 800 to produce the vapor stream.

FIGS. 3 and 4 illustrate the need for a vacuum distillation vessel toremove product from the fermentation broth. FIG. 3 shows the metaboliteprofile of a batch fermentation run. FIG. 3 shows that the biomass andethanol concentration increases exponentially during the initial phaseof the fermentation run. As the ethanol accumulates, exceeding aconcentration around 30 g/L, the biomass slows down due to the effectsof the ethanol on the microbes. This is further shown by FIG. 4, wherethe CO uptake and CO₂ production slows down around the same time thatthe ethanol concentration reaches around 30 g/L. This data illustratesthe needs for the vacuum distillation vessel of the current invention,where product concentration rates can be controlled to the point wherethe negative effects of product accumulation are mitigated and/orreduced.

The vacuum distillation vessel is capable of recycling product depletedfermentation broth to the bioreactor. The vacuum distillation vessel isdesigned to recover products, while ensuring the viability of themicroorganisms so that, when recycled, the microorganisms may fermentthe C1-containing gas in the bioreactor to produce products. FIGS. 5 and6 illustrate the ability of the vacuum distillation vessel to ensure theviability of the microorganisms from multiple variations of bioreactordesigns.

FIG. 5 shows the viability of microorganisms from a bioreactor with acertain configuration, where the fermentation broth is recycled from thevacuum distillation vessel to the bioreactor. The viability of themicroorganisms was measured at three times intervals from the bioreactor(Bioreactor 1) and from the vacuum distillation vessel (VD return). Asis shown in the graph, the viability of the microorganisms in the vacuumdistillation vessel is substantially equal to the viability of themicroorganisms in the bioreactor.

FIG. 6 shows the viability of the microorganisms from a bioreactor witha different configuration, where the fermentation broth is recycled fromthe vacuum distillation vessel to the bioreactor. The viability of themicroorganisms was measured at three times intervals from the bioreactor(Bioreactor 2) and from the vacuum distillation vessel (VD return). Asshown in the graph, the viability of the microorganisms in the vacuumdistillation vessel is substantially equal to the viability of themicroorganisms in the bioreactor.

FIG. 7 is a diagram showing the system according to one embodiment ofthe invention. The system is an energy optimized design 700 for acommercial sized plant, with minimal energy use and lowest carbonfootprint. Broth 702 comprising product from a fermentation process ispassed to degasser 704. The product exemplified is ethanol. Degasser 704may be a cyclonic entry atmospheric degasser. Vapor phase 706 may berouted to a scrubber of the fermentation process (not shown). Degassedbroth 708 is passed to separation vessel 710 at a location near to thetop of the vessel. Separation vessel 710 maybe a stripping column andmay be a vacuum stripper as described above. Product depleted broth 712is removed from vessel 710 at a location proximate to a total trap-outtray of vessel 710 and is passed to cooling tank 714. Cooling water instream 716 is indirectly heat exchanged in heat exchanger 718 to cool aportion of product depleted broth 720 removed from cooling tank 714generating cooled stream 719 which is passed to cooling tank 714.Another portion of product depleted broth 720 removed from cooling tank714 is passed to bioreactors of the fermentation process (not shown). Ableed stream 722 is removed from product depleted broth 720 removed fromcooling tank 714 and may be passed to wwtp (wastewater treatment plant)not shown. Boiler feed water 724 is passed into the bottom of vessel 710and purge stream 726 is removed from the bottom of vessel 710. Liquidwater stream 728 is also removed from the bottom of vessel 710 andpassed to reboiler 730 where liquid water stream is heated and returnedto vessel 710.

Product rich vapor stream 732 is removed as overhead from vessel 710.Product rich vapor stream 732 is compressed in multistage mechanicalvapor recompression unit 734 to generate compressed stream 736.Compressed stream 734 is passed to reboiler 730 and at least partiallycondensed thereby providing energy to drive reboiler 730 and heat liquidwater stream 728. Partially condensed stream 738 is passed to trimcondenser 740 and heat exchanged with cooling water stream 742.Non-condensed components removed in stream 744 and passed along withwater stream 748 to vacuum scrubber 746 where remaining product ethanolis scrubbed into stream 752. Non-condensable components not scrubbedinto the process water are removed as scrubber overhead stream 750. Trimcondenser 740 provides condensed stream comprising ethanol 754 which iscombined with stream 752 from scrubber 746 to form combined stream 756which in turn is passed to rectification column 760. In rectificationcolumn 760, product ethanol is distilled into overhead 762. Overhead 762is compressed in compressed in multistage mechanical vapor recompressionunit 766 to generate compressed stream 768 which is passed to heatexchanger 770 to heat rectification column liquid reboil stream 772.After heat exchange, stream 768 is passed to dehydration system 774 togenerate dry product stream 776. Optionally a portion of stream 768 maybe passed back to rectification column in stream 778 as reflux. Waterstream 780 is removed from the bottom of rectification column 760 andrecycled to the bottom of vessel 710. Through this recycle, water stream780 also directly provides heat to the bottom of vessel 710. Waterstream 780 may be indirectly heat exchanged with stream 756 in heatexchanger 758 to heat stream 756.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein. The reference to any prior art in this specification is not, andshould not be taken as, an acknowledgement that that prior art formspart of the common general knowledge in the field of endeavour in anycountry.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein. Variationsof those preferred embodiments may become apparent to those of ordinaryskill in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A process for removing a product from a fermentation broth, theprocess comprising: a. flowing a fermentation broth comprising microbialbiomass and product from a bioreactor to a broth stripper comprising atop, a bottom; b. partially vaporizing the fermentation broth in thebroth stripper to produce product rich vapor stream, and productdepleted liquid stream comprising live microbial biomass; c. passing theproduct depleted liquid stream back to the bioreactor and passing theproduct rich vapor stream to a trim condenser to generate a condensedproduct stream; d. passing the condensed product stream to arectification column comprising a top and a bottom, to separate arectification column overhead stream comprising product and arectification column bottoms stream; and e. passing the rectificationcolumn bottoms stream to the bottom of the broth stripper and directlytransferring heat from the bottom of the rectification column to thebottom of the broth stripper.
 2. The process of claim 1 wherein thecondensing of the product vapor stream is incomplete and a secondarycondenser is used to condense remaining vapor.
 3. The process of claim 2wherein the secondary condenser provides cooling through indirectcontact with cooling water or chilled water.
 4. The process of claim 1further comprising transferring heat, indirectly, by heat exchanging therectification column bottoms stream with the condensed product stream.5. The process of claim 1, wherein the fermentation broth is partiallyvaporised in the broth stripper at a temperature between 37° C. and 50°C. and a pressure between 40 mbar and 100 mbar.
 6. The process of claim1, wherein a viability of the microbial biomass in the product depletedliquid stream is greater than 85%.
 7. The process of claim 1, furthercomprising storing the product depleted liquid stream from the brothstripper in a cooled tank prior to passing to the bioreactor whereinwhere the cooled tank is temperature controlled to between 30° C. and37° C. and wherein the temperature control results in maintenance ofcell viability greater than 85%.
 8. The process of claim 1 wherein thebroth stripper comprises an associated broth stripper liquid reboiler,and the process further comprises a. compressing the product rich vaporstream; b. passing a liquid stream from the broth stripper to the brothstripper liquid reboiler and back to the broth stripper; and c.transferring heat, indirectly, from the compressed product rich vapor tothe liquid in the broth stripper liquid reboiler.
 9. The process ofclaim 2 wherein the product rich vapor stream is compressed to apressure of between 200 mbar to 300 mbar.
 10. The process of claim 2wherein the compressing is performed by multistage turbofans.
 11. Theprocess of claim 4 wherein between 2 and 4 turbofan stages are provided.12. The process of claim 2 wherein the broth stripper liquid reboiler ispositioned below theoretical distillation stages of the vessel which areseparated from an upper portion of the vessel by a total trap-out tray.13. The process of claim 2, wherein the transferring heat is a processin which the product rich vapor stream is indirectly contacted withliquid in the broth stripper liquid reboiler, resulting in condensationof the product rich vapor stream and evaporation of the liquid in theliquid reboiler.
 14. The process of claim 1 wherein the rectificationcolumn comprises an associated rectification column liquid reboiler, andthe process further comprises: a. compressing the rectification columnoverhead stream; b. passing a liquid stream from the rectificationcolumn to the rectification column liquid reboiler and back to therectification column; and c. transferring heat, indirectly, from thecompressed rectification column overhead stream to the liquid in therectification column liquid reboiler.
 15. The process of claim 1 furthercomprising passing the rectification column overhead stream to adehydration system.
 16. A system for removing a product from afermentation broth, the system comprising: a. a stripper vesselcomprising a feed inlet, a vapor outlet, a liquid outlet, a bottomsrecycle inlet, a reboiler liquid outlet, and a reboiler return inlet,wherein the feed inlet is in fluid communication with a bioreactor; b. astripper reboiler in fluid communication with the reboiler liquid outletand the reboiler return inlet; c. a first multistage mechanicalrecompression unit in fluid communication with the vapor outlet of thestripper vessel and the stripper reboiler; d. a trim condenser in fluidcommunication with the stripper reboiler; e. a rectification column influid communication with the trim condenser via a rectification columninlet conduit, the rectification column comprising an overhead outlet, abottoms outlet, a heat exchange outlet, and a heat exchange returninlet; f. a heat exchanger in fluid communication with the heat exchangeoutlet and the heat exchange return inlet; g. a second multistagemechanical recompression unit in fluid communication with the overheadoutlet of the rectification column and the heat exchanger; and h. abottoms conduit in fluid communication with the bottoms outlet of therectification column and the bottoms recycle inlet of the strippervessel.
 17. The system of claim 16 further comprising a dehydrationsystem in fluid communication with the heat exchanger and the secondmultistage mechanical recompression unit.
 18. The system of claim 16further comprising a bottoms stream heat exchanger in thermalcommunication with the bottoms conduit and the rectification columninlet conduit.
 19. The system of claim 16 further comprising a degasserin fluid communication with the bioreactor and the feed inlet of thestripper vessel.
 20. The system of claim 16 further comprising a coolingtank in fluid communication with the liquid outlet of the strippervessel.